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(Journal of Leukocyte Biology. 2003;73:574-583.)
© 2003 by Society for Leukocyte Biology

Toll-like receptor ligand links innate and adaptive immune responses by the production of heat-shock proteins

Udayasankar Kumaraguru, Christopher D. Pack and Barry T. Rouse

Department of Microbiology, University of Tennessee, Knoxville

Correspondence: Dr. Barry T. Rouse, Distinguished Professor of Microbiology, M409 Walters Life Sciences Building, University of Tennessee, Knoxville, TN 37996. E-mail: btr{at}utk.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The report shows that CpG can exert additional adjuvant effects by inducing cells that are normally inferior antigen (Ag)-presenting cells to participate in immune induction by cross-priming. Macrophages (M{phi}) exposed to protein Ag in the presence of bioactive CpG DNA released material that induced primary CD8+ T cell responses in DC-naïve T cell cultures. This cross-priming event was accompanied by up-regulation of the stress protein response as well as inflammatory cytokine expression in treated M{phi}. The material released was indicated to contain inducible heat shock protein-70 and epitope peptide, which in turn, were presented by dendritic cells (DCs) to responder T cells. Such an adjuvant effect by CpG may serve to salvage immunogenic material from otherwise inert depot cellular sites and additionally stimulate DCs to effectively cross-prime. The cross-priming, shown also to occur in vivo, may be particularly useful when Ag doses are low and have minimal opportunity for delivery to DCs for consequent direct priming.

Key Words: hsp70 • cross-priming • chaperone • DC • M{phi}


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid DNA (pDNA), encoding a wide range of antigens (Ag), represents a useful means of vaccination, being particularly effective at inducing T helper cell type 1-polarized CD4+T cell- and CD8+ T cell-mediated immunity [1 ]. It is not clear how pDNA immunization results in CD8+ T cell responses, but evidence exists for direct Ag presentation by plasmid-transfected Ag-presenting cells (APC) as well as cross-presentation [2 ]. In the latter instance, APC take-up exogenous Ag produced by other cell types and process it in a transporter associated with Ag processing (TAP)-dependent endogenous pathway [3 ]. This cross-presentation process may represent a crucial event. For example, immunization with stably transfected myoblasts, cells unable to pass on their transgene to other cells or to act themselves as APC, results in robust cytolytic T lymphocyte (CTL) responses [4 ]. Conceivably, cross-presentation becomes of greater relevance when pDNA immunization results in minimal delivery of pDNA to relevant APC such as dendritic cells (DCs).

Immunization with pDNA may be more effective at inducing CTL than immunization with proteins or inactivated vaccines, as the DNA itself may facilitate cross-presentation as well as exert adjuvant effects on APC. Accordingly, pDNA, as it contains unmethylated CpG dinucleotides that form motifs recognizable by pattern recognition receptors, activates several cell types involved in immunity [5 ]. These include macrophages (M{phi}), DCs, natural killer cells, and B cells, although not seemingly T cells [6 7 8 ]. The effects observed are multiple and include up-regulation of several key cytokines as well as accessory molecules involved in Ag presentation [9 , 10 ]. An additional effect of CpG, possibly explaining, in part, the potent, adjuvant effect of such motifs, could be their ability to facilitate Ag cross-presentation. Consequently, Ag expressed by nonprofessional APC could also become available for presentation by relevant APC to responder T cells. Support for the notion that CpG facilitates cross-presentation comes from the observation that immunization with CpG and protein or even peptide complexes results in potent CD8+ T cell responses [11 12 13 14 ]. Furthermore, some in vitro experiments have revealed that CpG protein treatment of immature DCs may "license" the cells to act as competent APC for CD8+ T cell responses [15 16 17 ]. Currently, it is not clear how CpG serve to license APC to use this alternative means of Ag presentation. It is also unclear, especially in vivo, what effect CpG has on the production and trafficking patterns of the Ag, which participates in cross-presentation.

Our previous studies on DNA immunization demonstrated that the source of cross-presenting Ag may be cells such as M{phi}, which readily take-up pDNA but do not themselves act as APC for CD8+T cell responses [18 ]. Experiments indicated that the transfected M{phi} produced material that included peptide bound to heat shock protein (hsp)70. This material, which was referred to as M{phi}-released factor (MRF), was taken up by DCs and primed CD8+ T cells. Accordingly, Ag may be generated by one or more cell types, and some are subsequently processed to a form suitable for cross-presentation by relevant APC such as DCs. Whereas prior studies have demonstrated that CpG can positively influence the cross-presentation efficiency of DCs [19 ], it is not clear if the efficiency by which cells produce the cross-presenting Ag is affected by CpG. In the present report, we have analyzed the influence of CpG on the production of select proteins implicated in mediating the cross-presentation process. Our results show a role for M{phi}–DC cooperation. Specifically, the M{phi} are primarily affected by the CpG, which results in the up-regulation of proinflammatory cytokines, stress proteins, and the uptake of the exogenous Ag. The degraded protein or peptide may be released in association with stress proteins [20 ], which in turn, shunts the material into the class I processing pathway of the DCs. The DCs, in addition to receiving the Agic material, also mature as a direct consequence of the CpG-mediated stimulus [19 ]. The combination of these two events results in efficient CTL priming.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Five- to six-week-old female C57BL/6 mice (H-2b) and BALB/c mice (H-2d) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). CD8-ovalbumin (OVA) transgenic (OT-1) was kindly provided by Dr. Michael Bevan (University of Washington, Seattle) [21 ]. The investigators adhered to the Guide for the Care and Use of Laboratory Animals, as proposed by the Committee on Care of Laboratory Animal Resources Commission on Life Sciences, National Research Council, in conducting the research described in this work. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care.

Proteins, peptides, CpG oligodeoxynucleotides (ODNs), and plasmid DNA-encoded Ag
Chicken egg albumin (OVA) grade VI was purchased from Sigma Chemical Co. (St. Louis, MO; cat. no. A2512). Herpes simplex virus (HSV) gB was provided by Chiron Corporation (Emeryville, CA). The H-2b-specific HSV peptide (aa 498–505; SSIEFARL) and chicken OVA peptide (aa 257–264; SIINFEKL) were synthesized and deblocked to provide free amino and carboxyl ends (Research Genetics, Birmingham, AL). CpG oligonucleotides, bioactive CpG-1826, nonbioactive CpG-1982, and methylated bioactive CpG-1845, were provided by Coley Pharmaceutical Group (Wellesley, MA) and possessed undetectable endotoxin levels. Fluorescein isothiocyanate (FITC) CpG and control CpG were kindly provided by Dr. Dennis Klinman (National Institutes of Health, Bethesda, MD). cDNA encoding the HSV-1 gene gB was inserted into the pcDNAI vector (Invitrogen, San Diego, CA), as described earlier [22 ]. cDNA encoding chicken OVA was obtained from Dr. Michael Bevan [23 ]. The purity and concentration of plasmid DNA, which was produced lipopolysaccharide (LPS)-free using endo-free columns obtained from Qiagen (Chatsworth, CA), were analyzed by A260 and A280 by agarose gel electrophoresis and ethidium bromide staining. Expression of the gB and OVA gene products was confirmed by Western blot analysis using commercially available antibodies (Ab) by transfection of splenic adherent cells from BALB/c mice in the presence of the cationic lipid lipofectamine (Invitrogen, Indianapolis, IN).

Ab
The following Ab were used: anti-hsp70, directed against a highly conserved 13-aa region near the N terminus of hsp70 [24 ]; normal rabbit serum (Zymed, San Francisco, CA; catalog number 01-6101); phycoerythrin (PE)-labeled anti-CD11b (PharMingen, San Diego, CA; catalog number 01715B) for M{phi}; PE-labeled anti-CD3 (PharMingen; catalog number 01085B) for T cells; FITC-labeled anti-B220 for B cells (PharMingen; catalog number 01125B); PE-labeled anti-CD11c (PharMingen; catalog number 09705B); PE-labeled anti-CD80 (PharMingen; catalog number 09605B); PE-labeled anti-CD86 (PharMingen; catalog number 09275B); and FITC-labeled anti-H-2b (PharMingen; catalog number 06044D) anti-hsp70 (Stressgen, Victoria, BC, Canada) and anti-hsp90 (Stressgen).

Isolation and purity of APC and responder T cells
DCs from splenocytes were isolated according to the procedure mentioned elsewhere [24 ]. Briefly, splenocytes were obtained from naive mice, and the cell concentration was adjusted to 2 x 107 cells in 3 ml RPMI-1640 medium containing 10% fetal calf serum (FCS; RPMI–10% FCS). These cells were overlayed onto 2 ml of a 14.5% metrizamide gradient column. After a low-speed centrifugation (200 g for 10 min), cells from the interface were collected and washed twice in RPMI–10% FCS. The pellet was resuspended in another 3 ml of the same medium, and the above procedure was repeated. Cells from the interface were collected. The purity of the preparation was checked by surface staining with monoclonal Ab (mAb) to 33D1 (kindly provided by Dr. Ralph Steinman, The Rockefeller University, New York, NY) and PE-labeled anti-CD11c. The maturation status of the DCs, analyzed by staining for major histocompatibility complex (MHC) class II and costimulatory molecule (CD80 and CD86) expression, indicated a heterogenous population.

The M{phi} were isolated as per the method described previously [25 ]. Briefly, the splenic cells were allowed to adhere onto a plastic tissue-culture T-150 flask for 90 min at 37°C. The nonadherent cells were processed for isolation of T cells described elsewhere. The adherent cell population was scraped off and allowed to readhere for another hour at 37°C. The readhered population was dislodged and resuspended in RPMI 1640 with 5% fetal bovine serum (FBS). Flow cytometric analysis was done after staining with mAb to CD11b to check for purity.

T cell isolation was done by separating B cells from the nonadherent population from above by passage through a nylon wool column and subsequently panning on anti-immunoglobulin (Ig)-coated plates. The separated cell population was analyzed for percentage of T lymphocytes by fluorescein-activated cell sorter (FACS) analysis (anti-CD3 staining) and was later used as responder naive T cells.

Intracellular staining
Following various treatment protocols, 2 x 106 cells were simultaneously fixed and permeabilized using 2 mL PermeaFix (OrthoDiagnostics, Raritan, NJ) for 40 min at room temperature (RT) in the dark, as described previously. Cells were then washed three times in phosphate-buffered saline (PBS). Nonspecific binding was inhibited by treating cells with Fc block for an additional 30 min at RT with gentle rocking. For the measurement of intracellular cytokine expression, cells were treated with anti-interleukin (IL)-6 PE or anti-mouse IL-1ß PE (1 µL/106 cells; PharMingen) for 40 min at RT in the dark. For measurement of stress proteins (hsp70 and hsp90), anti-hsp70 (Stressgen) and anti-hsp90 (Stressgen) were used. Cells were then washed twice in PBS and analyzed by flow cytometry. Flow cytometric analysis was performed on a FACScan with Cell Quest software (Becton Dickinson, San Jose, CA). Individual cells were gated on the basis of forward (FSC) and orthogonal scatter. The photomultiplier (PMT) for PE (FL2–height) was set on a logarithmic scale. Cell debris was excluded by raising the FSC–height PMT threshold. The flow rate was adjusted to <200 cells/s, and at least 10,000 cells were analyzed for each sample.

MRF production
M{phi} (5x105 cells/ml) were treated with OVA or gB protein in combination with CpG and alhydrogel (as described earlier in ref. [26 ]) for 24 h in 96-well, flat-bottom plates at 37°C. Following incubation for 24 h at 37°C and 5% CO2, 100 µl supernatant was harvested, passed through a 0.45-m filter, and added to 100 µl DC–T cell microculture [responder:stimulator (R:S) ratio of 10:1] in 96-well, U-bottom plates. Cultures were incubated at 37°C for 5 days. Cells were then pooled and used as effectors in a standard 4-h 51Cr release assay.

Confocal microscopy
Splenic adherent cells were stimulated with bioactive CpG (1826), nonbioactive (1982), methylated CpG (1845), and LPS for 6 h. The last 3 h of the incubation were performed in the presence of brefeldin A. Cells harvested after incubation were stained intracellularly for intracellular hsp70 (inducible) and analyzed by confocal microscopy to effectively visualize the intensity of staining for inducible (i)hsp70 at a single-cell level. The slides were visualized using a Leica TCS-4D confocal scanning microscope. Leica’s 3-D volume-rendering software was used to produce reconstructions of confocal stacks.

Cell-surface staining for M{phi} and DC
A portion of the isolated splenic populations was analyzed for cell-surface markers by flow cytometry to assess the purity of the preparations. The cells were blocked with heat-inactivated FBS and washed three times with FACS buffer (1xPBS with 1% bovine serum albumin and 0.05% NaN3). The cells were stained with mAb to 33D1 and CD11c for DCs, PE-labeled anti-CD11b for M{phi}, PE-labeled anti-CD3 for T cells, and FITC-labeled anti-B220 for B cells.

In vitro CTL induction
T cells (5x106 cells/ml) and APC (5x105 cells/ml) were cultured in 100 µl NCTC 109 and RPMI 1640 (1:1 v/v; Life Technologies, Gaithersburg, MD), supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 1 mM oxaloacetic acid, 0.2 U/ml bovine insulin, and 5 x 10-5 M 2-mercaptoethanol (2-ME) in 96-well, U-bottom plates to give a R:S ratio of 10:1. After 5 days, the cells were used as effectors in a standard 4-h 51Cr release assay.

M{phi} (5x105 cells/ml) were treated with purified pcDNAgB or pcDNA OVA (5–7 µg/ml) for 24 h in 96-well, flat-bottom plates at 37°C. Following incubation for 24 h at 37°C and 5% CO2, 100 µl supernatant was harvested, passed through a 0.45-m filter, and added to 100 µl DC–T cell microculture (R:S ratio of 10:1) in 96-well, U-bottom plates. Cultures were incubated at 37°C for 5 days and then pooled and used as effectors in a standard 4-h 51Cr release assay.

Cytotoxicity assay
Target cells (syngenic–MC-38; allogenic–EMT6) for peptide-specific lysis were prepared as follows: Target cells (2x106) were labeled in 500 µl RPMI 1640 with 100 µCi 51Cr for 90 min with appropriate peptides at a concentration of 7.5–10 µg/ml. For OVA-specific lysis, 2 x 106 EG7 were pulsed for 90 min with 100 µCi 51Cr in 500 µl RPMI 1640. After washing, 104-labeled targets and serial dilutions of effector cells were incubated in 200 µl RPMI 1640 with 10% heat-inactivated FCS in 96-well, V-bottom plates. The plates were centrifuged at 500 g for 3 min and incubated at 37°C and 5% CO2 for 4 h. A total of 100 µl supernatant fluid was collected, and radioactivity was measured using a gamma ({gamma}) counter (LKB). Specific cytotoxic activity was determined using the following formula: % Specific release = [(experimental release–spontaneous)/(total release–spontaneous release)] x 100, where experimental release is the radioactivity present in test samples, spontaneous release is the radioactivity of targets with the addition of media only, and the total release is the measure of activity after the addition of 3% Triton X. Each assay was performed in triplicate, and spontaneous release was less than 22% of total release by detergent in all assays.

Calculation of lytic units (LU)
A plot with percentage-specific lysis value versus the log of the effector cell number for each effector cell preparation was done. A lysis value of 20% was selected through which most of the declining titration curves passed. One LU was the number of lymphocytes required to yield 20% lysis. If the titration curve of an effector cell preparation failed to reach the selected lysis value (20%), the activity was referred to as < x LU, where "x" is the calculated minimum level.

Detection of Ag presentation using hybridoma
MRF, obtained using gB protein, was added to cultures containing DCs and HSV-2.3.2E2 [27 ] cells (CD8+ T cell hybridoma recognizing HSVgB498–505, kindly provided by Dr. Francis R. Carbone, The University of Melbourne, Victoria, Australia) for 36 h in 100 µl hybridoma media in 96-well, round-bottom plates (Falcon, Becton Dickinson). Culture supernate was then assayed for the presence of IL-2 by enzyme-linked immunosorbent assay (ELISA).

Blocking with anti-hsp Ab
The individual fractions of MRF were treated with polyclonal Ab to hsp104, gp96, hsp70, and hsp25 at a final concentration of 1:200, were incubated in a 37°C water bath for 45 min–1 h, and were then added to the DC–RF33.70 culture to test for blocking of MRF activity.

Nuclease treatment of MRF
The MRF was treated with 4 µl RNase-free DNase I (Sigma Chemical Co.; catalog number D4263) and 1 µl 10 mg/ml RNase A (Sigma Chemical Co.; catalog number R4875), incubated for 30 min at 37°C in a water bath and later analyzed for the presence of activity.

Purification of hsp70 peptide complex by adenosine 5'-diphosphate (ADP) affinity chromatography
The hsp70 was purified adapting the method of Peng et al. [28 ] with minor modifications. MRF (10 ml) obtained from various treatments was exchanged for buffer B [20 mM Tris-acetate, 20 mM NaCl, 15 mM 2-ME, 3 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.5] via gel filtration chromatography using Sephadex G-25. The sample was applied directly to an ADP–agarose column equilibrated with buffer B. The column was washed extensively with buffer B until protein was undetectable in the eluate by absorbance at 280 nm. Finally, the column was incubated with buffer B containing 3 mM ADP at RT for 30 min and subsequently eluted with the same buffer (25 ml). The buffer of the eluate was exchanged again with Sephadex G-25 for buffer C (20 mM Na2HPO4, 20 mM NaCl, pH 7.0). The protein was separated by anion exchange chromatography using MonoQ Sepharose and eluted over a 20–600 mM NaCl gradient. Fractions containing HSP70-phosphatidylcholine as a single protein, as determined by using the bioanalyzer and by Western blot with anti-HSP70 mAb, were pooled. Sephadex G-25, ADP agarose, MonoQ Sepharose, ADP, and all buffer ingredients were obtained from Sigma-Aldrich (St. Louis, MO). Protein content was determined by the Bradford assay using the protein assay kit of Bio-Rad (Bio-Rad Laboratories, Hercules, CA).

Western blot analysis
Proteins were extracted from cells following treatment with ice-cold RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 2.5% deoxycholate, 2 mM EGTA, 1 mM leupeptin, 1 mM aprotinin, 10 mM NaF, and 1 mM PMSF), and samples were cleaned by centrifugation at 15,000 g for 20 min at 4°C. For immunoprecipitation experiments, supernatant was carefully removed and incubated with primary Ab for 2 h on ice, and immunoprecipitates were collected with Protein A-Sepharose beads (Pharmacia Biotech, Piscataway, NJ) for 30 min at 4°C. The precipitate was washed three times with PBS and boiled in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer before electrophoresis on 10% SDS-polyacrylamide gels and were transferred to Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Bedford, MA). The membrane was blocked by incubation in Tris-buffered saline (TBS) buffer supplemented with 5% nonfat dry milk (Bio-Rad Laboratories) and 0.1% Tween-20 for 1 h at RT, washed three times in TBS buffer, and incubated overnight with appropriate primary Ab at 4°C. Membranes were washed three times with TBS buffer and were incubated with appropriate alkaline phosphate-conjugated secondary Ab for 1 h at RT. Detection of proteins was achieved by the enhanced chemiluminescence (ECL) system.

Inhibition of transport in DC
DCs were pretreated with brefeldin A (10 µg/ml) or monensin to inhibit Ag processing and presentation. DCs treated in this manner were unable to perform Ag processing and presentation.

M{phi} depletion
Dichloromethlene biphosphonate (Cl2MBP), entrapped in liposomes, provided by Van Rooijen and Sanders [29 ], was used to poison M{phi}. C57BL/6 mice were treated with 1.2 mg CL2MBP/mouse intravenously (i.v.) to eliminate M{phi} in the spleen. Splenocytes collected from Cl2MBP-treated mice analyzed at 3, 12, and 24 h post-treatment were processed for enumeration of DCs and M{phi} by staining with CD11c for DCs and F4/80 for M{phi}. After 24 h, M{phi} numbers were depleted by >90%, and hence, this time point was chosen for further studies. (Depletion was confirmed by FACS analysis with mAb F4/80.)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Production of cross-presenting signals
Previous studies have shown that M{phi} transfected with DNA-expression plasmids were ineffective APC [22 ], but such cells could release cross-presenting material that induced CD8+ T cell responses in DC–T cell cultures [18 ]. The cross-presenting material was indicated to include chaperone-bound peptides [22 ]. However, as is shown in Figure 1a and 1b , splenic M{phi} themselves pulsed with OVA protein, OVA DNA, or the target peptide SIINFEKL function poorly as APC (in comparison with DCs). Although M{phi} act as inferior APC when such cells were exposed to OVA DNA but not to OVA protein, material was released (herein referred to as MRF), which induced CTL responses in primary DC–T cell cultures. To determine if the difference in MRF stimulatory activity was associated with the CpG bioactivity of the plasmid DNA, M{phi} cultures were exposed to OVA protein (at various doses) along with an optimal amount of bioactive or control CpG. Although CpG plus protein alone induced minimal activity, the coincorporation of alhydrogel in the complex resulted in significant MRF production (Table 1 ). Whereas CpG-1826 induced 34 ± 8% lysis of EG-7 targets, nonbioactive (CpG 1982) and methylated bioactive CpG (1845) showed no activity. It has been suggested that the mechanism of action of repository adjuvants, such as aluminum salts, is primarily to act as a depot, thereby slowing the release of Ag [30 ]. However, in in vitro experiments, it probably facilitated CpG and protein to target the same M{phi}, resulting in activation and subsequent internalization of protein.



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  		Figure 1. DCs are more efficient than M{phi} in stimulating CD8+ T cells. (a) Splenic M{phi} or DCs, which were loaded osmotically with OVA protein, transfected with DNA encoding OVA protein, or pulsed with SIINFEKL peptide, were used as stimulators for responders (OT-1 cells). The R:S ratio varied from 10:1 to 0.62:1. The cells were incubated for 36–48 h at 37°C. The supernate collected from these cultures was analyzed for interferon-{gamma} (IFN-{gamma}) by ELISA. DCs and M{phi} without Ag (cells only) induced no detectable level of IFN-{gamma} (not shown). The difference observed between DCs and M{phi} was statistically significant (P<0.05). This figure shows the mean response of three separate experiments. Solid symbols, DCs; open symbols, M{phi}. (b) Splenic M{phi} or DCs were loaded osmotically with OVA protein and incubated overnight. They were then used at different ratios to stimulate OT-1 cells (36–48 h) to produce IFN-{gamma}. The last 6 h of the culture period was in the presence of brefeldin A (10 µg/ml). The cells collected from these cultures were then stained for intracellular IFN-{gamma}. The data were then analyzed using FACScan and Cellquest software. The shaded portions are the OT-1 cells stimulated but stained with isotype-control Ab. The overlay is stained with FITC-labeled anti-IFN-{gamma}. This figure represents the data obtained from one such experiment (DC/M{phi}:OT-1=1:2.5).

 

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  		Table 1. Release of CTL Generating Activity from M{phi} Exposed to Various Stimuli

 
A similar pattern of results was observed with glycoprotein B of HSV. Splenic M{phi} were pulsed with recombinant gB protein and CpG-1826 in the presence or absence of alhydrogel. Supernates obtained from CpG-1826 + gB + alhydrogel cultures induced primary in vitro CTL in naïve T cell–DC cultures (Fig. 2 ). The gB-specific CTL activity was 36 ± 5%. In comparison, gB alone pulsed culture was <5%. A similar pattern of results was observed when the MRF was assayed in a DC–gB-specific T cell hybridoma (2E2) culture system, and the readout was IL-2 production (CpG-1826+gB+alhydrogel=286±24 pg/ml; CpG-1982+gB+alhydrogel=17±4 pg/ml). Such results indicate that bioactive CpG stimulated M{phi} results in the uptake of exogenous Ag and the release of cross-presenting activity. In contrast, M{phi}, similarly treated with LPS, fixed Staphylococcus aureus–Cowans strain, Staphylococcus enterotoxin B (SEB), and Cholera toxin B subunit (CTB), in place of CpG, did not generate MRF, which could be cross-presented by DCs to naïve T cells (data not shown).



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  		Figure 2. M{phi} treated with CpG + alhydrogel + gB protein release Agic material that can be cross-presented by DCs, leading to priming of naïve T cells. M{phi} were treated with gB DNA, gB protein, gB protein+ CpG, and gB protein + CpG + alhydrogel (Alhy) for 18–24 h to induce the production of MRF. The released MRF was then analyzed for its ability to generate primary in vitro CTL after addition to naïve T cell–DC cultures. The CD8+ T cells generated were analyzed for their cytolytic activity against syngeneic targets. The values in the figure are averages of three separate experiments obtained with MC38 target cells previously infected with vaccinia virus encoding gB (VvgB inf.; ER-498–505). Also included but not shown are control targets (syngeneic-uninfected and allogeneic-infected) that yielded insignificant lysis.

 
M{phi} exposed to CpG up-regulate stress proteins involved in cross-presentation
Cross-presentation may involve a number of mechanisms, and chaperone-bound peptides may represent one such event [31 ]. To determine if M{phi} exposed to bioactive CpG up-regulate chaperones known to participate in cross-presentation, splenic adherent M{phi} were exposed for varying times to bioactive CpG, control ODNs (including methylated bioactive CpG and nonbioactive CpG), or LPS. Subsequently, cells were analyzed by FACS for the intracellular expression of various molecules or by immunoprecipitation of lysed cells and Western blot for ihsp70 expression. The results shown in Figure 3a reveal the intracellular expression of the cytokines IL-6 and IL-1ß as well as the two stress chaperones ihsp70 and hsp90. As is readily apparent, CpG-1826 induced hsp70, hsp90, as well as proinflammatory cytokines (IL-6 and IL-1ß). A small increase of these molecules was also observed with nonbioactive CpG 1982, but methylated CpG (1845) completely lacked such activity. (Similarly, methylation of plasmid DNA also resulted in loss of its immune-induction properties.) As shown in Figure 3b , the expression level of ihsp70 induced by CpG1826 was dose-dependent between 1 µg and 5 µg. Doses beyond 25 µg were toxic to the M{phi} culture.



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  		Figure 3. (a) Splenic adherent cells were stimulated with bioactive CpG (1826), nonbioactive CpG (1982), methylated CpG (1845), and LPS for 6 h. The last 3 h of the incubation were in the presence of brefeldin A. Cells harvested after incubation were stained intracellularly for hsp70 (inducible), hsp90, IL-6, and IL-1ß. The data were analyzed by flow cytometry using FACScan and Cellquest software. The figure rendition is a histogram overlay, and the shaded portions represent cells that were unstimulated. Controls that were included and not shown include cells stimulated as above but stained with isotype-control antibodies. The experiments were performed thrice with similar results. The figure is representative of one such experiment. (b) Splenic adherent cells were stimulated with bioactive CpG (1826) at varying doses ranging from 0 to 5 µg for 6 h. The last 3 h of the incubation were in the presence of brefeldin A. Cells harvested after incubation were stained intracellularly for hsp70 (inducible). The stained cells were analyzed by flow cytometry using FACScan and Cellquest software. The figure rendition is of histogram overlays, and the shaded portions represent unstimulated cells.

 
Hsp70 up-regulation following 1826 (bioactive CpG) treatment was also evident by confocal microscopy (not shown). To further ascertain if the MRF contained chaperone-peptide complexes, we purified the hsp70 peptide by ADP–affinity chromatography, described earlier by Peng et al. [28 ]. The purified material showed intense staining for hsp70 by Western blot analysis (Fig. 4 ) and in addition, induced a SSIEFARL peptide-specific response in vivo in B/6 mice (data not shown) when used as an immunogen. It is of interest to note that although LPS induced ihsp70, MRF was not generated by such treatment.



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  		Figure 4. ADP–affinity chromatography purification of MRF and detection of hsp70 complex by Western blot. MRF collected from splenic adherent cells treated with bioactive (1826), nonbioactive (1982), and methylated (1845) CpG and LPS or untreated were collected 24 h after stimulation. They were then passed through an ADP–agarose column. Few fractions (based on the protein content) collected were assessed for the presence of an hsp70–peptide complex. They were run on an SDS-PAGE gel and transferred to PVDF membrane, and hsp70 complex was revealed by ECL assay. Lane 1, Recombinant hsp70; lane 2, M{phi} alone; lane 3, CpG1982; lane 4, CpG1826; lane 5, CpG1845; lane 6, LPS; lane 7, recombinant hsp70.

 
Mechanism of MRF
It has been shown previously that antimalarials such as quinacrine negate the bioactive effect of CpG ODN but not the effect of LPS on M{phi} [32 ]. To ascertain the effect of quinacrine on CpG-induced ihsp70, M{phi} were pretreated with 0.01 µm, 0.1 µm, and 1 µm quinacrine and were then exposed to pretitrated amounts of CpG-1826. The cells were stained after incubation and then analyzed by FACS or confocal microscopy. The data presented in Figure 5 indicate that although 1 µm quinacrine abrogated the ihsp70 production in CpG-treated M{phi}, it had no effect on LPS-induced ihsp70 production. Results obtained from confocal microscopic screening (not shown) also supported this finding.



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  		Figure 5. Quinacrine pretreatment blocked CpG- but not LPS-induced hsp70. Splenic adherent cells pretreated with varying amounts of quinacrine were stimulated with bioactive CpG or LPS for 6 h. The last 3 h were in the presence of brefeldin A. The cells were then washed and stained intracellularly for inducible hsp70. Stained cells were analyzed by flow cytometry using FACScan and Cellquest software. The figure rendition is histogram overlays, the shaded portions depict cells not treated, and the unshaded portions depict treated cells. Additional control included cells stimulated as above but stained with isotype-control antibodies (not shown).

 
To further implicate the uptake of the CpG–protein complex as a necessary event for MRF production, M{phi} were pretreated with various doses of cytochalsin D to abrogate the function of the cytoskeleton. Such treated M{phi} failed to produce MRF (Table 2 ). In addition, treatment of MRF (that were collected from M{phi} pulsed with CpG+alhydrogel+OVA protein) with Ab to ihsp70 (0.01, 0.1, and 1 µg/ml) had a dose-dependent, inhibiting effect on the MRF activity (Table 2) . Taken together, the results indicate that the CpG-mediated effect on M{phi} requires the function of an active cytoskeleton for internalization and subsequent release of MRF. A major component of MRF activity is likely hsp70. In addition, the MRF activity as measured by CTL and T cell hybridoma activation was still present after treatment with nucleases (DNase and RNase). This indicates that the cross-priming activity was not attributable to the residual CpG activity on DCs directly.


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  		Table 2. Blocking with Anti-ihsp70 and Prevention of Cytoskeleton Activity in M{phi} Abrogate MRF Production

 
M{phi} depletion in vivo ablates the CpG–peptide-induced immune response
The above in vitro results indicate that CpG stimulation of Ag-exposed M{phi} induces them to generate and release cross-presenting material that is taken up by DCs and presented to CD8+ T cells. To establish if a similar event could occur in vivo, experiments were done in which the effect of CpG protein immunization was compared in normal animals with those in which M{phi} were depleted before immunization.

Accordingly, groups of mice were injected with FITC-labeled, bioactive CpG or control CpG and killed at 12-h intervals. Splenocytes were processed for flow cytometric analysis by staining with PE-labeled M{phi} (CD11b and F 4/80), DCs (CD11c), and B (CD22) and T (CD3) cell markers. In such experiments, a majority of cells with M{phi} markers were positive for FITC CpG, followed by CD11C + DC and B cells (Fig. 6 ). Having ascertained that M{phi} are the primary reservoir of CpG, we went on to measure the effect of M{phi} depletion on the response to CpG–Ag exposure. Animals were then pretreated with chlordonate liposomes or empty liposomes, and 24 h later, mice were injected i.p. with HSV–gB protein in combination with bioactive or control CpG. Subsequently, gB-specific CD8+ T cell responses were analyzed on day 6. As is evident in Table 3 , pretreatment of mice with Cl2MBP led to a significant reduction in the number of SSIEFARL (gB498-505)-specific T cells, as measured by tetramer (70% reduction) and intracellular staining for IFN-{gamma} (73% reduction). The CTL activity, as measured by 51Cr release assay, was also reduced by 83% in M{phi}-depleted mice compared with empty liposome-treated mice. Such data suggest that CpG increases the efficacy of immunization by making available cross-presenting Ag from cells, such as M{phi}, which are themselves inferior APC.



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  		Figure 6. M{phi} are the primary target of FITC CpG. Two groups of mice were injected intraperitoneally (i.p.) with FITC-labeled CpG or control CpG. Twenty-four hours later, spleen from these mice were removed and assayed by FACScan after staining them with a PE-labeled, cell-surface marker to distinguish them as DCs, M{phi}, and B and T cells. The figure shows the data for M{phi} (CD11b and F 4/80) and DC (CD11c) cells. Controls that were included but not shown are unstained, and isotype-control antibodies were stained. The experiment was repeated thrice with same pattern of results. The figure represents one such experiment.

 

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  		Table 3. M{phi} Depletion In Vivo Ablates CpG–Protein-Induced Immune Response

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These results reveal a possible mechanism by which CpG DNA achieves its remarkable adjuvanticity. Stimulatory effects on DCs, the principal APC for T cell activation, have been well documented (reviewed in ref. [33 ]). However, this report shows that CpG can exert additional adjuvant effects by inducing cells, which themselves are inferior APC, to participate in immune induction by cross-priming. Accordingly, M{phi} exposed to protein Ag in the presence of bioactive CpG DNA released material that induced primary CD8+ T cell responses in DC–naïve T cell cultures. This cross-priming event was accompanied by up-regulation of the stress-protein response as well as inflammatory cytokine expression in treated M{phi}. The material released was indicated to contain iHSP-70 and an epitope peptide, which in turn was presented by DCs to responder T cells. Such an adjuvant effect by CpG may serve to salvage immunogenic material from otherwise inert depot cellular sites. The cross-priming, shown also to occur in vivo, may be particularly useful when Ag doses are limiting for direct priming by DC.

A flurry of recent reports has documented the adjuvant function of CpG DNA, especially in rodents [34 35 36 37 38 39 ]. The effect is particularly striking on CD8+ T cell responses as well as on shifting the dominance of CD4+ T cell subset reactions [40 ]. Mechanistic studies have primarily emphasized the direct results of CpG DNA on DC function [19 ]. Such cells are activated by CpG to undergo maturation and express various costimulators and cytokines involved in effective Ag presentation [41 ]. The consequences of CpG–DNA exposure of cell types other than DC or lymphocytes have received minimal attention. However, Harding and co-workers [42 ] reported that CpG may serve to minimize the APC activity of M{phi}, at least under conditions of prolonged in vitro exposure. The present report documents a positive effect of CpG DNA on the immune-inducing function of M{phi}, and the effect is evident in vitro and in vivo. We show that M{phi} exposed to CpG DNA take-up proteins, such as OVA or gB HSV and subsequently, release material, herein referred to as MRF, induces DC naïve T cell cultures in turn to generate primary CTL. Previously, we had observed this cross-priming event only when M{phi} were exposed to expression plasmid DNA encoding gB or OVA [18 ]. In such studies, the activity of MRF was shown to depend on the function of an ATP-dependent hsp70, likely consisting of epitope peptide bound to the hsp70 chaperone [18 ].

The fact that M{phi} exposed to protein may also generate MRF, although only in the presence of bioactive CpG DNA, indicates that the adjuvant effect of plasmid DNA on M{phi} was a consequence of their CpG motif content. Furthermore, M{phi} exposed to CpG DNA were shown to up-regulate stress proteins, such as hsp70 and hsp90, as well as key cytokines likely involved in immune induction. However, absence of hsp90 deduction in our earlier report using plasmid-encoding proteins may be a result of the difference in the number of CpG motifs involved. Taken together, our results indicate that the adjuvant effect of CpG may be directed at cells, in addition to DCs, which are directly involved in Ag presentation. Presumably, the adjuvant effect of CpG is mediated via Toll-like receptor (TLR)9 signaling [43 , 44 ]. Although lacking appropriate knock-out animals to demonstrate the formal involvement of TLR9, the cross-priming event is likely to involve such key stimulation. The other TLR agonists and pathogen-associated molecular patterns investigated, such as LPS, SEB, and CTB, all failed to elicit M{phi} to generate MRF. The probable difference could be in their signal-transduction pathways. However, formal proof for the role of TLR as well as the potential involvement of other TLR receptors present on M{phi} require further evaluation. Such studies are now underway.

The notion that DNA vaccines induce immunity, at least in part via the process of cross-priming, has been evident for some time [45 ]. More recently, the Raz group has emphasized that CpG DNA causes DCs involved in Ag presentation to take-up exogenous Ag and process it in a TAP-dependent manner. This cross-presentation activity also involved IFN-{alpha}, which in an autologous manner, appeared responsible for the up-regulation of MHC proteins, TAP, and other costimulators involved in Ag presentation [19 ]. This view of CpG–DNA-aided cross-presentation of soluble Ag by DCs was also endorsed by a recent report from Maurer et al. [46 ]. In such studies, the adjuvant effect of APC appeared focused on the Ag-presenting DCs, the very cells also critical for immune induction by direct priming. Additionally, there exists evidence that hsp can exit mammalian cells and exert immunoregulatory effects [47 ] by interacting with specific receptors, especially those present on APC [48 , 49 ]. Such interaction induces maturation and migration [50 , 51 ] of these cell types, resulting in immune enhancement.

Our present study serves to emphasize that CpG motifs can function as an adjuvant by inducing cells that might take-up and potentially withhold Ag from immune recognition to generate and release such Agic material. Whether such a cross-priming process is unique to M{phi} or involves other potentially major sources of Ag, such as endothelial cells, fibroblasts, and myocytes [52 ], requires further investigation. We anticipate that other cells are likely involved and that this process of cross-priming may become particularly relevant in situations where DC uptake or expression of Ag is limited. Our preliminary in vivo studies indicate that cross-priming is not confined to in vitro culture systems. Thus, when M{phi} uptake of Ag was limited, as demonstrated in animals in which M{phi} function was compromised, the adjuvant effect of CpG on the CD8–T cell response to proteins was limited. We are currently extending such observations in an attempt to formally demonstrate the involvement of chaperone peptide cellular interactions in vivo, as well as to determine the involvement of molecules other than CpG that might mediate similar adjuvant effects at systemic and mucosal sites.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grants AI14981 and AI46462. We thank Dr. Arthur M. Krieg of Coley Pharmaceuticals for providing the CpG ODNs, Dr. Dennis Klinman (National Institutes of Health) for providing FITC CpG, Chiron Corporation for providing HSV–gB protein, Drs. Nico van Rooijen and Michael Bevan (University of Washington) for the OT-1 transgenic mice, and Dr. Francis R. Carbone (The University of Melbourne) for providing the 2E2 hybridoma.

Received September 24, 2002; revised January 21, 2003; accepted January 31, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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